Eukaryotic genomic DNA exists and functions in a highly compacted form, as a repeating array of nucleosomes called chromatin. Nucleosomes occlude much of the DNA that wraps them from interaction with other gene regulatory proteins and enzymes, yet nucleosomes actively help recruit other proteins to the vicinity of their wrapped DNA, through interaction of these other proteins with the nucleosomes' histone tail domains. Thus, the detailed locations of nucleosomes along the DNA may have both important inhibitory and facilitatory roles for chromosome function. This project is focused on understanding the principles that govern where nucleosomes are placed along the genome. In the current project period, we obtained data proving that genomes care where their nucleosomes are located, that genomes manifest this care through a DNA sequence code for nucleosome positioning, and that we are able partially to decipher this code. The nucleosome positions that we predict based only on our ability to read this code explain at least 50% of actual in vivo nucleosome positions. In the next grant period, we will pursue these findings in three key directions. Studies in Aim 1 will provide new data that will improve our histone-DNA affinity "profile", enabling it to better-predict nucleosome locations genome wide. Studies in Aim 2 will elucidate and experimentally evaluate the full competition between the constellation of site-specific DNA binding proteins and nucleosomes, when these molecules act together in their coupled dynamic equilibrium. Studies in Aim 3 will provide high resolution data on the distribution and nature of nucleosomes over divergent promoters genome-wide, and will apply these data together with a new experimental approach utilizing individual living cells, to elucidate the mechanism of promoter independence in divergently transcribed genes. Relevance: This project will elucidate the basic rules that govern where nucleosomes are placed along the genome. The detailed distribution of nucleosomes influences all aspects of chromosome function, including DNA replication, transcription, repair, recombination, and chromosome division. The understanding of eukaryotic genome structure that results from this work will help us understand, diagnose, and rationally design new kinds of therapies for, cancers and developmental and infectious diseases.